Neuropeptide Y ELISA Assay

Additional Information

Assay Principle

The Eagle Biosciences Neuropeptide Y ELISA Assay kit is an enzyme immunoassay for the quantification of Neuropeptide Y in serum, plasma, tissue and CSF extractions. This assay employs the competitive enzyme immunoassay technique. A secondary antibody has been pre-coated onto a microtiter plate and non-specific binding sites were blocked. Fc regions of primary antibodies specific for target peptides can bind to the secondary antibodies on microtiter plate. The Fab regions of primary antibodies are competitively bound by biotinylated peptide and targeted peptides in samples. After washing away any unbound substances, the antibody bound to the solid phase is detected by using TMB as a substrate. The reaction is monitored at 450 nm±2 nm. Quantification of unknown samples is achieved by comparing their absorbance with a reference curve prepared with known standards. The intensity of color development is inversely proportional to the amount of Neuropeptide Y in the samples.

Assay Procedure

1. Add 50 μl of 1X assay buffer as Total Binding. Empty wells should be left as blank.
2. Add 50 μl of prediluted peptide standards, 50 μl positive controls and 50 μl samples into corresponding wells. It is advisable to assay each condition in duplicates.
3. Add 25 μl of primary antibody into each well except the Blank wells.
4. Add 25 μl of Biotinylated peptide into each well except the Blank wells. It is not recommended to use a multi-channel pipette to load the primary antibody and biotinylated peptide.
5. Seal the microtiter plate with acetate plate sealer (APS). Incubate for 2 hours at RT. Orbital shaking at 300-400 rpm is recommended.
6. Centrifuge Streptavidin-HRP vial and pipette 12 μl of Streptavidin-HRP into 12ml of 1X Assay buffer. Vortex thoroughly. Prepare fresh.
7. Remove APS from plate.
8. Aspirate each well and wash, repeating the process 1 times for a total 2 washes. Wash by filling each well with 1× Assay Buffer (350 μl) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating, decanting or blotting against clean paper towels.
9. Add 100 μl Streptavidin-HRP solution into each well.
10. Reseal the plate with APS. Incubate for 1 hour at RT. Orbital shaking at 300-400 rpm is recommended.
11. Remove APS from plate.
12. Wash as according to step 8.
13. Add 100 μl TMB substrate solution into each well.
14. Reseal the plate with APS. Incubate for 1 hour at RT. Orbital shaking at 300-400 rpm is recommended. (Protect from light)
15. Remove APS from plate.
16. Add 100 μl 2N HCl into all wells to stop the reaction.
17. Read the OD with a microplate reader at 450 nm immediately.

Typical Standard Curve